µPhos: a scalable and sensitive platform for functional phosphoproteomics

Mass spectrometry has revolutionized cell signaling research by vastly simplifying the identification and quantification of many thousands of phosphorylation sites in the human proteome. Defining the cellular response to internal or external perturbations in space and time is crucial for further illuminating functionality of the phosphoproteome. Here we describe µPhos, an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture experiments in < 8 hours total processing time. By minimizing transfer steps and reducing liquid volumes to < 200 µL, we demonstrate increased sensitivity, over 90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify more than 20,000 unique phosphopeptides in a human cancer cell line using 20 µg starting material, and confidently localize > 5,000 phosphorylation sites from 5 µg. This depth covers key intracellular signaling pathways, rendering sample-limited applications and extensive perturbation experiments with hundreds of samples viable. Abstract Figure